ESPE2022 Poster Category 1 Adrenals and HPA Axis (52 abstracts)
Background: The blood-brain barrier (BBB) is a multicellular neurovascular unit (NVU) that allows selective passage of necessary molecules into the central nervous system (CNS) while limiting the entry of neurotoxins and most drugs. A cross talk with pericytes and neural cells mediates the acquisition of tight junctions (TJs) in brain microvascular endothelial cells (BMECs), which limit the paracellular passage of solutes, thereby regulating CNS homeostasis. However, the mechanisms by which NVU cells communicate to mediate TJ formation remains a mystery. Retinoic acid (RA), a key signaling molecule during vertebrate embryonic development, is commonly used to enhance functional barrier properties in in vitro BBB models. However, its physiological relevance and affected pathways are not fully understood. P450 oxidoreductase (POR) acts as an electron donor for microsomal cytochromes, thereby regulating their enzymatic activity.
Aim: To study the role of POR in the functional development of BBB properties.
Methods: We generated iPSC-derived brain microvascular endothelial-like cells (iBMECs) derived from both, POR-deficient (PORD)patients and CRISPR/Cas9-mediated POR-depleted isogenic lines (PORM), to study the role of POR in BMEC acquirement of functional barrier properties.
Results: We demonstrate that POR is expressed at the BBB and in iBMECs and is enzymatically active in control iBMECs while severely attenuated in PORD iBMECs. An analysis for the TJ relevant markers ZO-1, GLUT-1 and CLDN-5 showed a control-like expression of ZO-1, GLUT-1 and CLDN-5 in PORD iBMECs. PORM iBMECs exhibited non-continuous expression of ZO-1, decreased expression of GLUT-1 and no detectable CLDN-5. Studies on iPSC differentiation to iBMECs with a gradient of RA concentrations demonstrate that PORD and PORM iBMECs fail to develop functional barrier properties under higher RA concentrations and suggest that POR is necessary for the RA-dependent acquisition of TJs and barrier functions. We tested RA-dependent changes in gene expression within each cell line by calculating the differentially expressed genes (DEGs) as the fold change of each RA concentration compared to iBMECs differentiated without RA. DEGs were then further filtered by their base means. Control iBMECs showed a gradual dose-dependent increase in RA-induced number of DEGs. Contrarily, PORD iBMECs showed an increased number of DEGs that was apparent already at the lower RA concentrations. This was further accentuated in the PORM iBMECs.
Conclusion: We demonstrate that POR regulates RA homeostasis, and that POR-deficiency leads to accumulation of RA within iBMECs, resulting with impaired expression of TJs and consequently to dysfunctional development of barrier properties.
15 Sep 2022 - 17 Sep 2022